Method of Making Perfluorocarbon Emulsions With Non-Fluorinated Surfactants

ABSTRACT

The present invention provides a method of making a FC emulsion. The method comprises mixing an FC immiscible hydrophilic liquid and a solid emulsifying agent by agitation at a temperature elevated above the phase transition temperature of the emulsifying agent and below the boiling temperature of the FC immiscible hydrophilic liquid, and adding FC to the mixture of step (a) and agitating at the elevated temperature to disperse droplets of FC in the FC immiscible hydrophilic liquid to form the FC emulsion. The invention also provides another method of making an FC emulsion, which does not require a solid emulsifying agent. The method comprises mixing an FC immiscible hydrophilic liquid and an emulsifying agent to form a first mixture; mixing a stabilizing agent with the first mixture to form a second mixture; and mixing FC with the second mixture to form a third mixture to disperse droplets of FC in the FC immiscible hydrophilic liquid and to form the FC emulsion, wherein the stabilizing agent reduces ability of the droplets to move within a continuous phase of the FC emulsion.

CROSS-REFERENCE TO THE RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/253,572, filed Sep. 24, 2002, which is incorporated herein byreference.

FIELD OF THE INVENTION

The invention relates generally to stable emulsions capable ofgas-supersaturation, and methods of their preparation and use.Specifically, the present invention relates to stable fluorocarbonemulsions prepared with non-fluorinated emulsifying and stabilizingagents and methods of their preparation utilizing conventional mixingequipment.

BACKGROUND OF THE INVENTION

Fluorocarbons (fluorine substituted hydrocarbons) and perfluorocarbons(fluorocarbons in which all of the hydrogen atoms have been replacedwith fluorine) have numerous applications in the biomedical fieldbecause of their unique chemical and biological properties. Thesecompounds are clear, colorless, odorless, nonflammable, biocompatible,and have low reactivity. In addition, they are capable of havingdissolved in them large amounts of gases, including oxygen, carbondioxide, and air, per unit volume (WO 96/40057). Accordingly,fluorocarbons (FCs) and perfluorocarbons (PFCs) have been successfullyused as carriers in applications wherein oxygen must be supplied toorgans and tissues.

For example, it has been demonstrated that liquid ventilation with PFCsquickly and efficiently returns arterial oxygen and carbon dioxide bloodgas pressures to normal physiological values in premature lambssuffering from respiratory distress syndrome (H.-J. Lehmler et al.,Chemtech, 1999, v. 29, No. 10, 7-12). Aqueous emulsions of PFCs havebeen explored as artificial blood substitutes or as perfusion fluids forthe preservation of internal organs to be transplanted (GB 1,549,038).Some FC blood substitutes, such as FLUOSOL® (Green Cross Corporation,Osaka, Japan) and OXYGENT® (Alliance Pharmaceutical Corp., San Diego,Calif.) have been clinically evaluated. A synthetic cerebrospinal fluidcomprising an oxygenated FC emulsion has been used to deliver oxygen toischemic neurologic tissue (U.S. Pat. No. 4,758,431).

Compositions of FCs may also be used for the treatment of wounds, forexample, burns, as described in U.S. Pat. No. 4,366,169. The patentteaches contacting a wound with a liquid fluorocarbon directly, orindirectly through a dressing, such as a sponge, gauze, foam, dispersionor gel. Recently, the application of FC emulsions in cosmetics anddermatology has been suggested. It is believed that by delivering oxygeninto the skin, FC emulsions may improve skin metabolism, impede skinaging, and protect skin from injurious environmental effects (Oxynoid,O. E. et al., Art. Cells, Blood Subs., and Immob. Biotech., 1994, 22(4),1331-1336).

Fluorocarbons are extremely hydrophobic and are not miscible with mostorganic or aqueous systems. Therefore, typically, FCs and PFCs are usedin the forms of aqueous emulsions, dispersions, and gels. The stabilityof the FC emulsions, dispersions, and gels is a key concern in theirbiomedical use (U.S. Pat. No. 6,113,919).

The dispersed phase of fluorocarbon emulsions must have a stableparticle size to be suitable for biomedical use. One of the shortcomingsof existing emulsions is their low stability. The particle size of acommercially available fluorocarbon emulsion, such as FLUOSOL, forexample, can be maintained only if it is transported and stored in afrozen state. The frozen emulsion is then defrosted and mixed with annexsolutions before use. These storage requirements seriously limit thefield of application of such fluorocarbon emulsions. Although morestable fluorocarbon emulsions are being developed, it is desirable tohave fluorocarbon emulsions that are stable enough to store for longperiods without refrigeration and in various packaging configurationsfor such applications, such as topical, spray, ointment, etc.

Fluorocarbons are oily substances that are immiscible with water, andtherefore, fluorocarbon-in-water emulsions are generally prepared byusing emulsifying agents, such as lecithins and/or poloxamers, such asPluronic-F-68, to disperse the fluorocarbon and stabilize the emulsion.However, most conventional emulsifying agents have a low affinity forfluorocarbons.

Several strategies in preparing stable FC emulsions have been exploredby researchers. One approach involves dispersing very small FC droplets,less than 0.3 μm, preferably 20-200 nm in diameter, in a continuousphase. This approach is particularly advantageous in blood substituteapplications because emulsions with larger droplet sizes can bedangerous in intravenous use (GB 1,549,038; U.S. Pat. No. 4,865, 836).However, since the gas dissolved in a larger droplet may be releasedover a more extended period of time, stable emulsions comprising largerFC droplets are desirable to achieve a maximum therapeutic effect intopical applications.

A surfactant (or emulsifying agent) may be used to stabilize thedispersed droplets through the electrostatic and steric repulsion of thesurfactant molecules that surround each dispersed PFC or FC droplet. Forexample, U.S. Pat. Nos. 4,569,784 and 4,879,062 describe a stable gel ofFC comprising up to 10% (wt/wt) emulsifier. The FC gel is prepared by amulti-step procedure comprising preparing an FC emulsion, concentratingthe FC emulsion by a high-speed centrifugation, and removing excessfluid by a separation process. In this method, the type and the quantityof the surfactant used is not crucial, as long as it provides effectiveemulsification. However, this method involves a complex multi-stepprocedure and requires high pressure homogenization or sonically-inducedcavitations to effectively disperse fluorocarbons in water.

Another approach to improving the stability of fluorocarbon emulsions isto develop more effective surfactants, for example, those which arefluorophilic. Fluorinated surfactants have been shown to improve thestability of FC emulsions (U.S. Pat. No. 6,113,919) However, this classof surfactants has also been shown to have a negative environmentalimpact that led to the withdrawal of some previously commerciallyavailable fluorinated surfactants.

U.S. Pat. Nos. 5,573,757 and 6,113,919 focus on the further improvementof the available surfactant systems. A stable FC emulsion is prepared byutilizing a combination of a surfactant and a fluorophilic/lipophyliccompound, which act together in association to stabilize the emulsion bysurrounding the dispersed droplets and preventing their coalescence.However, the environmental concerns associated with such fluorinatedcompounds have greatly limited their use and availability in recentyears.

Another strategy is to prepare microemulsions, i.e., preparations ofcompounds which organize themselves spontaneously into dispersed systems(U.S. Pat. No. 3,778,381; FR A 2 515 198). Microemulsions, while stablethermodynamically, require a substantial amount of surfactants in theirformulations, which may lead to bioincompatibiltiy for medicalapplications. In an example of a microemulsion described by Cecutti etal., Eur. J. Med. Chem., 24, 485-492 (1989), the dispersed phase isitself totally constituted of mixed hydrocarbon/fluorocarbon molecules.Thermodynamically stable microemulsions, when broken, can be restored bya mild agitation. U.S. Pat. No. 4,722,904 teaches a PFC microemulsioncomprising a system of two surfactants, the primary surfactant beingnon-ionic and water-soluble, and the secondary surfactant beinghydrotropic and present in an amount sufficient to disorder anywater-primary surfactant gels. However, it is not clear whether suchmicroemulsions are suitable for biomedical and cosmetic applications. Sofar, PFC microemulsions have been used only as reference liquid for thecalibration and/or quality control of blood gas analyzers (U.S. Pat.Nos. 4,151,108 and 4,722,904) and their safety for intravenous use hasbeen questioned (U.S. Pat. No. 6,113,919).

U.S. Pat. No. 5,637,318 describes a dermatological agent containing anemulsion of asymmetrical lamellar aggregates comprising FC surrounded byat least three layers of phospholipid molecules. The dermatologicalagent is prepared using an ultrasonic disintegrator and the stability ofthe emulsion is achieved by limiting the size of the lamellar aggregatesto 50-1000 nm, with a mean diameter of 244 nm.

Therefore, a number of conventional FC emulsions, dispersions, and gelsare prepared by multi-step complex methods that utilize a high-pressureapparatus or ultrasound to form an emulsion and require furtherconcentration by centrifugation. Other FC emulsions are stabilized withfluorinated surfactants that cause certain environmental concerns or byforming microemulsions that have not been used in biomedicalapplications to date. Still another group of FC emulsions limit the sizeof the FC droplets and do not provide an optimal therapeutic effect intopical applications.

SUMMARY OF THE INVENTION

In view of the above-noted shortcomings of the conventional FC emulsionsand methods of their preparation, it is an object of the presentinvention to provide stable FC emulsions using non-fluorinatedemulsifying agents. It is also an object of the present invention toprovide simple and low energy methods for preparing stable FC emulsionsand gels by utilizing conventional mixing equipment. It is a furtherobject of the present invention to provide methods that allow thepreparation of stable FC emulsions with a broad range of FC dropletsizes.

These and other objects are achieved in the present invention by using astabilizing agent that reduces the ability of the FC droplets to movewithin the continuous phase. Conventionally, the FC emulsions arestabilized either by decreasing the size of the dispersed FC dropletsand/or by selecting a surfactant that surrounds the fluorocarbondroplets reducing interfacial surface tension and causing electrostaticand steric repulsion between the droplets, inhibiting their coalescence.The present invention, on the other hand, stabilizes the FC emulsion byfurther decreasing the ability of the dispersed FC droplets to movewithin the continuous phase. This result is achieved by several meansincluding using a stabilizing agent to alter the physical properties ofthe continuous phase, an emulsifying agent, and a method of making theemulsion that result in a highly stabilized fluorocarbon emulsion.

Accordingly, one aspect of the present invention is directed to afluorocarbon (FC) emulsion, comprising:

a continuous FC immiscible hydrophilic liquid phase; and

a dispersed phase comprising FC suspended as droplets within thecontinuous phase;

the emulsion further comprising an emulsifying agent and a stabilizingagent, wherein the stabilizing agent reduces the ability of the FCdroplets to move within the continuous phase.

The FC may be selected from the group consisting of linear, branched andcyclic fluorinated hydrocarbon compounds, derivatives and mixturesthereof. The FC may be a PFC. In one embodiment, the PFC isperfluorodecalin. The PFC may also be substituted, such as with bromineor iodine. If substituted, the perfluorocarbon may be perfluorooctylbromide or perfluorooctyl iodine.

The stabilizing agent may be selected from a group consisting of cetylalcohol, stearyl alcohol, behenyl alcohol, glyceryl stearate,polyoxyethylated fatty acid (PEG-75 stearate), polyethylene glycol etherof cetyl alcohol (ceteth-20), polyethylene glycol ether of stearylalcohol (steareth-20), hydrogenated phosphotidylcholine, and mixturesthereof. The amount of the stabilizing agent may be in the range fromabout 0.05% to about 10% (wt/wt). In another embodiment, both thestabilizing agent and the emulsifying agent are hydrogenatedphosphotidyl choline.

The continuous FC immiscible hydrophilic liquid phase of the presentinvention may further comprise a thickening agent to increase theviscosity of the emulsion. In one embodiment, the thickening agent isselected from the group consisting of carboxylic acid polymers,polyacrylamide, polysaccharides, and gums.

Another aspect of the present invention is directed to a method ofmaking an FC emulsion. The method comprises:

(a) mixing an FC immiscible hydrophilic liquid and a solid emulsifyingagent by agitation at a temperature elevated above the phase transitiontemperature of the emulsifying agent and below the boiling temperatureof the FC immiscible hydrophilic liquid; and

(b) adding FC to the mixture of step (a) and agitating at the elevatedtemperature to disperse droplets of FC in the FC immiscible hydrophilicliquid and to form the FC emulsion.

The FC immiscible hydrophilic liquid of the present invention can beglycerin, an aqueous solution or other liquid that is hydrophilic andnot miscible with the FC. The aqueous solution is water or a solutioncomprising water and components soluble or dispersable in water,including stabilizing agents, humectants or other additives that mayprovide desirable characteristics to the finished emulsion.

Preferably, the emulsifying agent is a non-fluorinated compound. In oneembodiment, the non-fluorinated emulsifying agent is a hydrogenatedphospholipid. The hydrogenated phospholipid may be selected from thegroup consisting of hydrogenated phosphatidylcholine,lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphanolipids, phosphatidic acid, and mixturesthereof.

The elevated temperature is in the range from about 40° C. to about 90°C. The above-described method may further comprise a step of cooling theobtained emulsion to form a viscous solution or gel.

The instant invention also provides another method of making an FCemulsion, which does not require a solid emulsifying agent. The methodcomprises:

(a) mixing an FC immiscible hydrophilic liquid and an emulsifying agentto form a first mixture;

(b) mixing a stabilizing agent with the first mixture to form a secondmixture; and

(c) mixing FC with the second mixture to form a third mixture todisperse droplets of FC in the FC immiscible hydrophilic liquid and toform the FC emulsion, wherein the stabilizing agent reduces the abilityof the droplets to move within a continuous phase of the FC emulsion.

A further aspect of the present invention is directed to an oxygendelivery agent for the delivery of oxygen into oxygen-depletedenvironments. The agent comprises the FC emulsion described above,wherein the FC emulsion is saturated or supersaturated with oxygen. Inone embodiment, the oxygen delivery agent contains at least about one mlof oxygen at standard temperature and pressure (STP) per one ml of FCemulsion.

The present invention also provides a method of preparing an oxygendelivery agent. The method comprises:

(a) providing the FC emulsion described above; and

(b) exposing the FC emulsion to oxygen under conditions sufficient tooxygenate the emulsion to a predetermined degree.

The emulsion may be oxygenated by exposing it to oxygen gas underatmospheric pressure or under high-pressure (above atmospheric pressure)conditions. In one embodiment, an oxygenated emulsion with a pO₂ of upto 10,000 mm Hg may be obtained by exposing the emulsion to oxygen gasat 180 psig for one hour.

The present invention offers many economical and technical advantagesover the conventional methods. The present invention provides stable FCemulsions, dispersions, and gels of varying FC contents and drop sizesusing non-fluorinated emulsifying agents and conventional mixingequipment. The visco-elastic properties of the FC preparations of thepresent invention range from low viscosity emulsions/dispersions tohighly visco-elastic and semi-solid gels. Also, the FC emulsions of thepresent invention are capable of dissolving large quantities of gases,such as oxygen and carbon dioxide. Finally, the disclosed methods ofpreparing FC emulsions involve conventional mixing and do not requirefurther concentrating steps. Accordingly, the methods of the presentinvention are fast, simple, and inexpensive. Since only standardlaboratory equipment is utilized, no specialized training of laboratorypersonnel is required.

Oxygenated emulsions of the present invention are well-suited to be usedin applications in which local delivery of oxygen are desirable. Forexample, they may be used in topical applications for the healing ofwounds, bums, and bruises. The oxygenated emulsions may also beincorporated into pharmaceutical products containing antibiotics,nutritive elements, hydrating agents, and other beneficial andtherapeutic substances, for optimal therapeutic and healing effect. Inaddition, the oxygenated emulsions of the present invention may beincorporated into various cosmetic products, such as creams, ointments,masks, and exfoliates, to name a few.

The above-mentioned and other features of this invention and the mannerof obtaining them will become more apparent, and will be bestunderstood, by reference to the following description, taken inconjunction with the accompanying drawings. These drawings depict only atypical embodiment of the invention and do not therefore limit itsscope.

DESCRIPTION OF THE FIGURES

FIG. 1 schematically represents a production process for oxygen deliveryagent in accordance with one embodiment of the present invention.

FIG. 2 schematically represents an oxygen delivery agent prepared inaccordance with an embodiment the present invention.

FIG. 3 shows partial oxygen pressure (pO₂) of porcine skin as a functionof time in an in vitro model.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of the present invention, the following terms aredefined as follows:

Fluorocarbon is a fluorinated hydrocarbon in which at least one hydrogenatom is replaced by a fluorine atom.

Perfluorocarbon—a substantially fluorinated or completely fluorinatedmaterial which is generally, but not necessarily, a liquid at ambienttemperature and pressure and which has the ability to transport gases,such as oxygen and carbon dioxide. “Substantially fluorinated” in thisspecification means that most of the hydrogen atoms of a compound havebeen replaced by fluorine atoms, such that further replacement does notsubstantially increase the gas transport capability of the material. Itis believed that this level is reached when at least about 80-90% of thehydrogen atoms have been replaced by fluorine atoms (U.S. Pat. No.4,569,784).

Emulsion—a system consisting of a liquid dispersed with or without anemulsifier in an immiscible liquid in a form of stable (non-coalescing)droplets or microdroplets.

Continuous phase—the carrier phase of an emulsion, wherein the fluidelements are in continuous contact and not isolated from one another.

Dispersed phase—the phase of an emulsion, wherein the fluid elements arediscrete and isolated from one another, as in the form of droplets ormicrodroplets, or contained within micelles.

Thickening agent—a chemical agent having the property that, when addedto a liquid mixture, increases its viscosity, or resistance to flow.

Viscous solution—a solution with viscosity greater than about 50 cp, andwhich may or may not be dependent upon applied shear and/or temperature.

Gel—a semisolid that behaves like a solid at rest. Gels may flow athigher shear rates due to their shear thinning properties. The viscosityof gels may range from a thickened pourable liquid gel to a soft solidstick.

Liquid Crystals—orderly planes of oil and water separated by layers ofsurfactant. Under certain conditions, a combination of oil, water andsurfactant will, instead of forming micelles, result in the formation ofsuch liquid crystals. The liquid crystals may co-exist with regularemulsions. The presence of liquid crystals enhances the stability of theemulsion due to the formation of a covering “skin” around the dropletsand also due to the formation of a three-dimensional network through thecontinuous phase. The covering “skin” prevents the coalescence ofdroplets. A layered structure also reduces the van der Waals attractionbetween the droplets, inhibiting coalescence. The three-dimensionalnetwork reduces the ability of the droplets to move within thecontinuous phase (for more information, please refer to “Liquid Crystalsand Emulsions” in Emulsions and Solubilization, Shinoda K and Friberg S,John Wiley & Sons, Inc. (1986) and Jansson, P. and Friberg, S.;Molecular Crystals and Liquid Crystals, 34:75 (1976)).

In its first aspect, this invention provides an FC emulsion, comprising:

a continuous FC immiscible hydrophilic liquid phase; and

a dispersed phase comprising FC suspended as droplets within thecontinuous phase;

the emulsion further comprising an emulsifying agent and a stabilizingagent, wherein the stabilizing agent reduces the ability of the FCdroplets to move or coalesce within the continuous phase.

The continuous FC immiscible hydrophilic liquid phase of the FC emulsionof the present invention may include an aqueous phase and otherwater-miscible substances, such as glycerin, propylene glycol,short-chain alcohols, and other hydrophilic liquids. Such hydrophilicliquids mix readily with water and may be added individually or incombination to the continuous phase.

The water-immiscible dispersed phase of the FC emulsion consists of FCsor derivatives thereof. FCs are chosen for their inert chemistry andhigh oxygen solubility (about twenty times greater than that of water).Substantially, any FC may be used. Suitable FCs include, but are notlimited to, linear, branched and cyclic fluorinated hydrocarboncompounds, derivatives and mixtures thereof. In one embodiment, FC maybe a perfluorocarbon (PFC). Examples of acceptable PFCs include, but arenot limited to, C6-C9 perfluoroalkanes, perfluoroperhydrofluoranthrene,perfluorodecalin, perfluoroperhydrophenanthrene,bis(perfluor-hexyl)-1,2-ethene, perfluoro-1,3-dimethylcyclohexane,perfluoromethyldecalin, perfluoroisopropyldecalin, a mixture ofperfluorodixylylmethane and perfluorodixylylethane, and a mixture ofperfluoroperhydrophenanthrene and perfluoro n-butyl decalin.

Hydrogen atoms of the FC may be substituted with a halogen, such as Br,Cl, or I, in addition to fluorine. In one embodiment, the PFC isperfluorooctyl bromide or perfluorooctyl iodine. In another embodiment,a commercial product PFC 5080 (a product of 3M, St. Paul, Minn.) isused. PFC 5080 is a mixture of perfluorocarbons containing predominatelyperfluorooctane.

The FC emulsions of the present invention may contain FCs in the amountfrom about 2% to about 90% (wt/wt), preferably from about 10% to about70% (wt/wt). However, FC concentrations outside of the above ranges mayalso be used.

Because FCs are oily compounds that are immiscible with water, anemulsifying agent is typically used to prepare an FC-in-water emulsion.The emulsifying agent assists in dispersing FC and stabilizing theemulsion. Any emulsifying agents capable of dispersing FC may be used.However, due to environmental concerns, preferably, the emulsifyingagent is a non-fluorinated compound. In biomedical and cosmeticapplications, additionally, the emulsifying agent is preferablybiocompatible (non-irritating) and should result in an emulsion have asufficient shelf life. A shelf life of at least six months, preferablygreater than one year, is generally considered sufficient.

Examples of such biocompatible, non-fluorinated compounds include, butare not limited to, a block copolymer of ethylene oxide and propyleneoxide, phospholipids, and a polyoxyethylene derivative of a fatty acidester of sorbitan. In one embodiment, the block copolymer of ethyleneoxide and propylene oxide is a triblock copolymer of ethylene andpropylene oxide with an average molecular weight of 7680 to 9510 and 81wt % oxyethylene (poloxamer 188) or PLURONIC® F-68 (BASF Corporation,Mount Olive, N.J.). In another embodiment, the emulsifying agent ispolyoxyethylene 20 sorbitan monostearate (polysorbate 60).

In still another embodiment, a hydrogenated phospholipid is used. Thehydrogenated phospholipid may be selected from the group consisting ofhydrogenated phosphatidylcholine, lysophosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphanolipids, phosphatidic acid, and mixtures thereof. It isanticipated that other emulsifying agents having ahydrophilic-lipophilic balance (HLB) or a combined HLB value similar tothat of polysorbate 60 and PLURONIC® F-68 may be used in the preparationof the FC emulsion of the present invention. In a preferred embodiment,the hydrogenated phospholipid is hydrogenated phosphatidylcholine.

Typically, the emulsifying agent is used in an amount in the range fromabout 1% to about 15% (wt/wt). In one embodiment, the amount of theemulsifying agent is in the range from about 3% to about 7% (wt/wt).However, greater amounts may be used if desired.

As explained in the Introduction section, conventional FC emulsions arestabilized either by decreasing the size of the dispersed FC dropletsand/or by selecting a surfactant that surrounds the fluorocarbondroplets and reduces the interfacial surface tension and causingelectrostatic and steric repulsion between the droplets, inhibitingtheir coalescence.

Surfactants are commonly amphiphilic molecules having a hydrophobic tailregion and hydrophilic head region. However, FCs are water-immisciblesubstances that are neither hydrophilic nor lipophilic. Thus, theinterfacial surface tension between FCs and an FC immiscible hydrophilicliquid phase cannot be effectively reduced by conventional surfactantsalone. Accordingly, U.S. Pat. No. 6,113,919 teaches the use of acombination of a conventional surfactant and a fluorophilic/lipophilicmolecule, which act together in association to reduce the interfacialsurface tension and stabilize the emulsion.

This invention takes a different approach that does not require the useof fluorophilic/lipophilic compounds. Instead, in this invention, the FCemulsion is unexpectedly stabilized by decreasing the ability of thedispersed FC droplets and micelles to move within the continuous phasealtogether. As a result, the FC droplets cannot combine with each otherand destabilize the emulsion. It is a discovery of the present inventionthat the mobility of FC droplets may be decreased by adding astabilizing agent to the continuous phase to alter the physicalproperties of that phase.

Accordingly, the stabilizing agent of the present invention is acompound that reduces the ability of the FC droplets to move within thecontinuous phase. The stabilizing agent may be selected from the groupconsisting of cetyl alcohol, stearyl alcohol, behenyl alcohol, glycerylstearate, polyoxyethylated fatty acid (PEG-75 stearate), polyethyleneglycol ether of cetyl alcohol (ceteth-20), polyethylene glycol ether ofstearyl alcohol (steareth-20), hydrogenated phosphotidylcholine, andmixtures thereof.

Typically, the amount of the stabilizing agent is in the range fromabout 0.05% to about 20% (wt/wt). Preferably, the amount of thestabilizing agent is in the range from about 0.5% to about 5% (wt/wt).

In one embodiment, hydrogenated phosphotidyl choline is used as both thestabilizing agent and the emulsifying agent.

The FC emulsion of the present invention may be in a form of a viscoussolution, liquid crystal, or gel. In one embodiment, the continuousaqueous phase further comprises a thickening agent to increase theviscosity of the emulsion. Preferably, the thickening agent is added insuch an amount that the viscosity of the FC emulsion is at least about50 centipoise under normal conditions.

The thickening agent may be selected from the group consisting of the FCemulsion of claim 24, wherein the thickening agent is selected from thegroup consisting of carboxylic acid polymers, polyacrylamide,polysaccharides, and gums. The carboxylic acid polymers may behomopolymers of acrylic acid cross-linked with allyl ethers of sucroseor pentaerytritol (carbomers) or copolymers of C10-30 alkyl acrylateswith one or more monomers of acrylic acid or methacrylic acidcross-linked with allyl ethers of sucrose or pentaerytritol(acrylates/C10-30 alkyl acrylate crosspolymers).

The polysaccharides may be cellulose and cellulose derivatives. In oneembodiment, the polysaccharides are selected from the group consistingof hydroxyethyl cellulose, carboxymethyl hydroxyethyl cellulose,microcrystalline cellulose, cetyl hydroxyethyl cellulose, hyaluronicacid, chitosan, dextran, and derivatives thereof.

The gums may be selected from the group consisting of agars, gelatin,guar gum, and algin.

As explained above, it is an unexpected discovery of the presentinvention that the mobility of the FC particles within the continuousphase may be decreased and the FC emulsion may be stabilized by adding astabilizing agent to the continuous phase. Accordingly, another aspectof the present invention is directed to methods of making an FCemulsion. The method comprises:

(a) mixing an FC immiscible hydrophilic liquid and an emulsifying agentto form a first mixture;

(b) mixing a stabilizing agent with the first mixture to form a secondmixture; and

(c) mixing FC with the second mixture to form a third mixture todisperse droplets of FC in the FC immiscible hydrophilic liquid and toform the FC emulsion, wherein the stabilizing agent reduces the abilityof the droplets to move within a continuous phase of the FC emulsion.

It is also an unexpected discovery of the present invention thatmechanical agitation of the heated ingredients, including a solidemulsifying agent, forms a stable suspension of FC droplets within thecontinuous phase. The FC emulsion obtained in accordance with the abovemethod is thermally and mechanically stable and will not separate intoits constituent phases unless subjected to extreme conditions.

Accordingly, the present invention also provides another method ofmaking an FC emulsion. This method comprises:

(a) mixing an FC immiscible hydrophilic liquid and a solid emulsifyingagent by agitation at a temperature elevated above the phase transitiontemperature of the emulsifying agent and below the boiling temperatureof the FC immiscible hydrophilic liquid; and

(b) adding FC to the mixture of step (a) and agitating at the elevatedtemperature to disperse droplets of FC in the FC immiscible hydrophilicliquid and to form the FC emulsion.

The mixing and agitation may be conducted by any conventional means, forexample, by hand stirring, aeration, propeller agitation, turbineagitation, colloid milling, homogenizing, high-frequency or ultrasonicoscillation (sonication), micro-fluidization and the like. In oneembodiment, a homogenizer is used.

Due to environmental concerns, the emulsifying agent used in eithermethod is preferably a non-fluorinated compound. The emulsifying agentmay be selected from the group consisting of a block copolymer ofethylene oxide and propylene oxide, phospholipids, and a polyoxyethylenederivative of a fatty acid ester of sorbitan. In one embodiment, theblock copolymer of ethylene oxide and propylene oxide is a triblockcopolymer of ethylene and propylene oxide with an average molecularweight of 7680 to 9510 and 81 wt % oxyethylene (poloxamer 188 orPLURONIC® F-68). In another embodiment, the emulsifying agent ispolyoxyethylene 20 sorbitan monostearate (polysorbate 60).

When a solid emulsifying agent is required, it is preferably ahydrogenated phospholipid. The hydrogenated phospholipid may be selectedfrom the group consisting of hydrogenated phosphatidylcholine,lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphanolipids, phosphatidic acid, and mixturesthereof. In one embodiment, the hydrogenated phospholipid is ahydrogenated phosphotidyl choline.

Typically, the amount of the emulsifying agent is in the range fromabout 1% to about 15% (wt/wt), preferably, in the range from about 3% toabout 7% (wt/wt).

Typically, the elevated temperature is in the range from about 40° C. toabout 90° C.

This method may further comprise a step of cooling the obtained emulsionto form a viscous solution or gel. In one embodiment, the viscosity ofthe viscous solution is greater than the viscosities of plasma andblood, which are in the range of I to 6 centipoise. In anotherembodiment, the viscosity is at least about 50 centipoise.

In one embodiment, the FC emulsion is cooled to at least about 40° C.

The above-described method of making FC emulsion may further comprise astep of processing the viscous solution to reduce the size of the FCdroplets. Such processing step may be conducted by homogenization orother suitable methods known to those skilled in the art.

Depending on the type of the intended use of the FC emulsion of thepresent invention, step (a) of the above method may further compriseadmixing formulation additives into water. Such formulation additivesare well known to those skilled in the art and may be selected from agroup consisting of stabilizing agents, thickening agents,preservatives, humectants, emollients, colorants, pH adjustment agents,buffers, texture-enhancing compounds, sunscreens, antioxidants,chelating agents, fragrances, flavorings, processing aids, and otherbioactive or inert cosmetic and pharmaceutical ingredients.

For example, the stabilizing agent may be selected from a groupconsisting of cetyl alcohol, stearyl alcohol, behenyl alcohol, glycerylstearate, polyoxyethylated fatty acid (PEG-75 stearate), polyethyleneglycol ether of cetyl alcohol (ceteth-20), polyethylene glycol ether ofstearyl alcohol (steareth-20), hydrogenated phosphotidylcholine, andmixtures thereof. The amount of the stabilizing agent may be in therange from about 0.05% to about 20% (wt/wt), preferably, from about 0.5%to about 5% (wt/wt). In one embodiment, both the stabilizing agent andthe emulsifying agent are hydrogenated phosphotidyl choline. Thehumectants are substances that are added into formulations to preservethe moisture content of the materials to which they are added, slowevaporation and enhance moisturization. The humectants are well known inthe art and may be selected from the group consisting of glycerin,propylene glycol, butylene glycol, sorbitol and others. Other additivesare also well known in the art and may be may be selected from the groupconsisting of thixotropic, whitening agents and processing aids.Thixotropic agents are added generally to alter the flowcharacteristics, or rheology, of the emulsion.

In another aspect of the present invention, the emulsion may be treatedto contain more gas, preferably oxygen, than would result under normalconditions. Accordingly, the present invention provides a gas deliveryagent for the delivery of gas into gas-depleted environments. The agentcomprises the FC emulsion described above, wherein the FC emulsion issaturated with a gas. When the gas is oxygen, the concentration ofoxygen in the FC emulsion of the present invention is at least about 0.3ml of oxygen (STP) per one ml of FC emulsion at 1 atmosphere. Thepartial pressure of oxygen, or pO₂, in the emulsion is generally above760 mm Hg in these embodiments

The gas delivery agent of the present invention may be supersaturatedwith gas. For example, in one embodiment in which the gas is oxygen, theconcentration of oxygen in the emulsion is at least about 1 ml of oxygen(STP) per one ml of FC emulsion. In another embodiment, theconcentration of oxygen in the emulsion is at least about 2 ml of oxygen(STP) per ml of FC emulsion. The partial pressure of oxygen, or pO₂, inthe emulsion is generally above 10,000 mm Hg in these embodiments, andcan be as high as 11,000 mm Hg or higher.

The present invention also provides a method of preparing a gas deliveryagent. The method comprises:

(a) providing the FC emulsion of the present invention; and

(b) exposing the FC emulsion to a gas under conditions sufficient togasify the emulsion to a predetermined degree.

The FC emulsion may be exposed to gas under atmospheric pressure orunder a pressure that is above atmospheric pressure. For example, whenthe gas is oxygen, the emulsion may be exposed to oxygen under 180 psifor sufficient time to achieve pO₂ in the emulsion of at least about10,000 mm Hg.

In one embodiment, illustrated in FIG. 1, the ingredients of the FCemulsion are heated and mixed in a mixer 1 and the obtained emulsion ispumped by pump 2 into a pressurized oxygenation vessel 3. Mechanicalagitation is performed in this unit with dual pitched blades to ensurerapid equilibration with oxygen to produce an oxygen delivery agent.Lastly, the internal pressure of the oxygenation vessel 3 forces theoxygen delivery agent into the filling station 4 for bottling.

Because the oxygen delivery agent of the present invention is capable ofdelivering high levels of oxygen into the skin tissues, it has manybiomedical and cosmetic applications. For example, it may be used intopical applications for the healing of wounds, burns, and bruises. Theoxygen delivery agent may also be incorporated into pharmaceuticalproducts containing antibiotics, nutritive elements, hydrating agents,and other beneficial and therapeutic substances, for optimal therapeuticand healing effect. The oxygen delivery agent may be applied directly tothe skin or may be incorporated into a dressing for application to theskin. Examples of such dressings include, but are not limited to, gauze,bandages, and other materials suitable for maintaining the oxygendelivery agent in contact with the skin. Application of an occlusivewound dressing inhibits diffusive transport of oxygen to the ambientenvironment and ensures maximal transport of oxygen to the treatmentsite.

The oxygen delivery agent may also be incorporated into various cosmeticproducts, such as creams, ointments, masks, and exfoliates, to name afew. In one embodiment, the oxygen delivery agent of the presentinvention delivers oxygen to intact skin in cosmetic applications toimprove skin texture and tone. For example, the oxygen delivery agentmay be incorporated into a skin care cream or a cosmetic mask. Suchproducts may be applied by a skilled skin care professional in theoffice or by a consumer at home.

The FC emulsion or gas delivery agent of the present invention ispreferably stored under pressure in a dispensing bottle to maintain thephase equilibria between the solubilized gas in the FC emulsion and thepressurized gas and to prevent gas dissolution and escape from theemulsion. When the gas is oxygen, oxygen gas may also serve as thevehicle propellant in such a delivery system, or, an indirectpropellant, such as another gas, for example, nitrogen, applied to theoutside of a barrier within the dispensing bottle, can be employed.

Preferably, the pressurized container holding the gas delivery agentshould maintain an internal pressure that, at a minimum, remains equalto or greater than the equivalent dissolved gas partial pressure. Also,in a preferred embodiment, the pressurized container allows fulldispensation of the gas delivery agent (greater than 95% of totalcharging weight dispensed).

The following examples are intended to illustrate, but not to limit, thescope of the invention. While such examples are typical of those thatmight be used, other procedures known to those skilled in the art mayalternatively be utilized. Indeed, those of ordinary skill in the artcan readily envision and produce further embodiments, based on theteachings herein, without undue experimentation.

EXAMPLES Example 1

Preparation of the FC emulsion Ingredients % (wt/wt) A Water 42 BGlycerin 12 C Hydrogenated Phospholipid 4.5 D Cetyl alcohol 0.6 EStearyl alcohol 0.6 F Polysorbate 60 0.5 G Perfluorodecalin 39.5 HTocopheryl acetate 0.4A is mixed into B. The mixture is heated to 70° C. in a reaction vesselwith agitation. When the temperature reaches 70° C., C is added to themixture at 70° C. with agitation. An overhead stirrer is used to conductthis step. After C is uniformly dispersed in the mixture, D, E and F areadded with continued agitation. When all of the above ingredients areuniformly dispersed, G is added slowly at a controlled rate withagitation. When all G has been added, H is added while maintaining thetemperature at 70° C. The mixture is cooled slowly to room temperature.Alternatively, prior to cooling, the mixture may be subjected tohomogenization and then cooled slowly to room temperature.

Example 2

Preparation of alternate FC emulsion Ingredients % (wt/wt) A Water 42.5B Propylene Glycol 30 C Hydrogenated Phospholipid 4.4 D Cetyl alcohol0.3 E Stearyl alcohol 0.3 F Polysorbate 60 0.2 G Glyceryl stearate,PEG-75 stearate, 0.8 ceteth-20, steareth-20 (Emulium Delta, Gattefosse,France) H Perfluorodecalin 20 I Tocopheryl acetate 0.5 J Dimethicone 0.7K Benzyl alcohol 0.4 L Methyl paraben 0.1 M Propyl paraben 0.05 NHyaluronic acid 1A is mixed into B. The mixture is heated to 70° C. in a reaction vesselwith agitation. When the temperature reaches 70° C., C is added to themixture at 70° C. with agitation. An overhead stirrer is used to conductthis step. After C is uniformly dispersed in the mixture, D, E, F and Gare added with continued agitation. When all of the above ingredientsare uniformly dispersed, H is added slowly at a controlled rate withagitation. When all H has been added, I and J are added whilemaintaining the temperature at 70° C. The mixture is cooled slowly toroom temperature. While the mixture is cooling, K, L, M and N are added.Alternatively, prior to cooling, the mixture may be subjected tohomogenization and then cooled slowly to room temperature.

Example 3

Preparation of alternate FC emulsion Ingredients % (wt/wt) A Water 48 BPropylene Glycol 34 C Hydrogenated Phospholipid 4 D Cetyl alcohol 0.4 EStearyl alcohol 0.4 F Polysorbate 60 0.3 G Glyceryl stearate, PEG-75stearate, 1 ceteth-20, steareth-20 (Emulium Delta, Gattefosse, France) HPerfluorodecalin 10 I Tocopheryl acetate 0.8 J Dimethicone 0.5 K Benzylalcohol 0.4 L Methyl paraben 0.1 M Propyl paraben 0.05A is mixed into B. The mixture is heated to 70° C. in a reaction vesselwith agitation. When the temperature reaches 70° C., C is added to themixture at 70° C. with agitation. An overhead stirrer is used to conductthis step. After C is uniformly dispersed in the mixture, D, E, F and Gare added with continued agitation. When all of the above ingredientsare uniformly dispersed, H is added slowly at a controlled rate withagitation. When all H has been added, I and J are added whilemaintaining the temperature at 70° C. The mixture is cooled slowly toroom temperature. While the mixture is cooling, K, L and M are added.Alternatively, prior to cooling, the mixture may be subjected tohomogenization and then cooled slowly to room temperature.

Example 4 Preparation of the Oxygen Delivery Agent

The FC emulsion prepared in accordance with any of Examples 1 through 3is charged with 180 psi of oxygen in a pressurized mixer for 90 minutes.The obtained oxygen delivery agent is packaged in a pressurizedcanister, containing approximately 1.8 ml of oxygen (STP) per ml FC. Aschematic representation of the oxygen delivery agent is shown in FIG.2.

Example 5 Oxyyen Transfer in Freshly Harvested Porcine Skin Tissue

A needle oxygen measuring device (FO₂TON Measuring System, Ocean Optics,Inc.), calibrated for temperature, was used to measure tissue oxygenlevels that penetrated approximately 1 mm thick pig skin sections on thebench.

Freshly harvested porcine skin was shaved and cut to a thickness ofapproximately 1 mm, leaving the epidermis intact. The oxygen deliveryagent of Example 2 was applied to the external surface of two skinsections (Sample 1 and Sample 2), while a direct-contact pO₂ probe wasapplied to the opposite side.

FIG. 3 shows partial oxygen pressure (pO₂) of porcine skin as a functionof time over a period of approximately 90 minutes. The continuousmeasurements showed an elevation of transdermal oxygen levels toapproximately 200 mm Hg, with the increase occurring over a 30-minuteperiod and sustained for at least 1 full hour. However, because of theslaughterhouse treatment of the skin (blanching) that generally impedesabsorption/convection of oxygen, better oxygen transfer is expected infresh skin specimens.

1. A method of making a fluorocarbon (FC) emulsion, comprising: (a)mixing an FC immiscible hydrophilic liquid and a solid emulsifying agentby agitation at a temperature elevated above the phase transitiontemperature of the emulsifying agent and below the boiling temperatureof the FC immiscible hydrophilic liquid; and (b) adding FC to themixture of step (a) and agitating at the elevated temperature todisperse droplets of FC in the FC immiscible hydrophilic liquid and toform the FC emulsion.
 2. The method of claim 1, wherein the elevatedtemperature is in the range from about 40° C. to about 90° C.
 3. Themethod of claim 1, further comprising a step of cooling the obtainedemulsion.
 4. The method of claim 3, wherein the emulsion is cooled to atleast about 40° C.
 5. The method of claim 1, further comprising a stepof processing the emulsion to reduce the size of the FC droplets.
 6. Themethod of claim 5, wherein the processing step comprises homogenization.7. The method of claim 1, wherein step (a) further comprises admixingformulation additives into the FC immiscible hydrophilic liquid.
 8. Themethod of claim 7, wherein the formulation additives are selected fromthe group consisting of stabilizing agents, thickening agents,preservatives, humectants, emollients, colorants, pH adjustment agents,buffers, texture-enhancing compounds, sunscreens, antioxidants,chelating agents, fragrances, flavorings, processing aids, and otherbioactive or inert cosmetic and pharmaceutical ingredients.
 9. An FCemulsion prepared in accordance with the method of claim
 1. 10. A methodof making an FC emulsion, comprising: (a) mixing an FC immisciblehydrophilic liquid and an emulsifying agent to form a first mixture; (b)mixing a stabilizing agent with the first mixture to form a secondmixture; and (c) mixing FC with the second mixture to form a thirdmixture to disperse droplets of FC in the FC immiscible hydrophilicliquid and to form the FC emulsion, wherein the stabilizing agentreduces ability of the droplets to move within a continuous phase of theFC emulsion.
 11. The method of claim 10, wherein step (c) furthercomprises admixing formulation additives into the FC immisciblehydrophilic liquid.
 12. The method of claim 11, wherein the formulationadditives are selected from a group consisting of stabilizing agents,thickening agents, preservatives, humectants, emollients, colorants, pHadjustment agents, buffers, texture-enhancing compounds, sunscreens,antioxidants, chelating agents, fragrances, flavorings, processing aids,and other bioactive or inert cosmetic and pharmaceutical ingredients.13. An FC emulsion prepared in accordance with the method of claim 10.